MICAL-1 Protein Redox-CH domains (6xHis-tag)

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MICAL (MIC01) is an intracellular flavoprotein monoxygenase, conserved from insects to mammals, that functions as a catalyst for oxidation-reduction (redox) reactions. Terman's group showed that MICAL interacts with F-actin and uses NADPH as a cofactor to oxidize actin at Met44 and Met47. As part of the MOXtrue product line, Human MICAL1 protein (MIC01) accession # Q8TDZ2 (MICA1_HUMAN) has been produced and purified from a bacterial expression system. The recombinant protein is N-terminally 6x Histidine tagged and is comprised of the Redox and CH domains (aa: 1-612) of the full length protein. Validation studies have shown that MIC01 can oxidize actin which will reduce subtilisin A cleavage by >90% in limited proteolysis assays. MIC01 has also been validated for the production of oxidized actin which can be used in downstream applications such as sedimentation assays and actin polymerization assays.
Each lot of purified protein is quality controlled to provide high batch to batch consistency, see COA documents.
MICAL-1 (MIC01) Protein Purity Determination
A 10 ?g sample of MICAL-1 protein was sepa-rated by electrophoresis in a 4- 20% Tris-Glycine gel and stained with Coomassie Blue. Protein quantitation was performed using the Precision RedTM Protein Assay Reagent (Cat.# ADV02). Mark12 standard molecular weight markers are from Invitrogen.
Subtilisin Assay on MsrB2 Treated Oxidized Actin
MICAL-oxidized actin (MetO Actin) (Cat# MXA95) was diluted to 0.1 mg/ml (2.3 ?M) and left untreated, or treated with DTT (10 mM), MB201 (0.3 ?M) , or a combination of both (see method). 2 ?g of each sample was then left untreated, or treated with subtilisin (1:200 w/w) for 15 min. Samples were then separated by SDS-PAGE and visualized with Coomassie staining.
Actin Sedimentation +/- MICAL-1 Treatment
Actin (Cat# AKL99) was diluted to 0.2 mg/ml (4.6?M) or 0.8 mg/ml (18.4 ?M) (see method). Samples were then incubated with 2x polymerization buffer or 2x polymerization buffer supplemented with MIC01 + NADPH for 1 h at room temperatire. Samples were spun in an ultracentrifuge at 100, 000 g for 1.5 h. Samples were then separated by SDS-PAGE and visualized with Coomassie staining.
Actin Polymerization +/- MICAL-1 Treatment.
Pyrene labeled-actin (Cat. #AP05) was diluted to 0.1 mg/ml (2.3 ?M) (see method). Samples were then incubated with 2x polymerization buffer supplemented with nothing (labeled Control), NADPH alone (labeled +NADPH), MIC01 alone (labeled +MIC01), or the combination of both (labeled +Both). Upon actin polymerization fluorescence was detected with a spectrophotometer (see method below). A.U. = arbitrary units
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